Identify potential liability issues

in early stages of drug discovery

www.wuxiapptec.com

Enabling Unbounded Possibilities

Drug induced toxicity is one of the leading causes of drug candidate failure in preclinical and clinical testing stage, and

also the major reason for the withdrawal of approved drugs from the market. Initial toxicity testing is required during the

nonclinical phase of development and relies primarily on animal studies. While these animal models have provided useful

information on the safety of chemicals, they are relatively expensive, low-throughput, and sometimes inconsistently

predictive of human biology and pathophysiology because of the species difference. With increasing numbers of new

chemical entities for environmental and pharmaceutical uses, it is necessary to find a rapid and efficient method to

screen chemicals for their potential toxicities. Since most drug-induced toxicity is due to toxic effects at the cellular level,

alternative in vitro models are increasingly being used to estimate in vivo responses, to reduce and/or replace in vivo

animal testing, and to increase the throughput. This idea is supported by the 2007 NRC report, "Toxicity Testing in the

21st Century (TT21C): A Vision and a Strategy1." This report predicts substantial advances in toxicity testing in the near

future which are much more specific and predictive of human toxicity. In vitro toxicity testing studies are faster, simpler

and more scalable so they can be used in the early drug discovery stage to predict potential risk. This would not only be

economical but ethical as it could markedly reduce the number of animal usage.

To address the need for early stage in vitro toxicity testing, WuXi AppTec Biology offers a panel of toxicity assays at the

cellular level by utilizing cutting-edge technologies, such as conventional and automated patch clamp and high content

screening (HCS). Applying these assays to your lead ID and optimization strategy can help provide a more thorough

analysis of the severity and specificity of toxicity. This information can then be used to guide candidate compounds

through the planning and execution of downstream in vivo tests.

1) Cardiotoxicity

• hERG and other ion channel test

• Stem cell derived cardiomyocyte test on MEA

2) General Cytotoxicity

• Cell Viability Assay

• Apoptosis Assay

• CellTox™ Green Cytotoxicity Assay

• ApoTox-Glo™ Triplex Assay

3) Mitochondrial Toxicity

• Mitochondrial Membrane Potential Assay

• Mitochondrial Reactive Oxygen Species (ROS) Assay

• Oxygen Consumption Assay: MitoXpress® Xtra OCR Assay

• MitoXpress® Cellular Energy Flux OCR/ECAR Assay

• Glucose/Galactose Assay

• MitoBiogenesis Assay

4) Lipotoxicity

• Phospholipidosis Assay

• Steatosis Assay

5) Hepatotoxicity

6) Nephrotoxicity

7) Genotoxicity

• Ames Assay

• In vitro Micronucleus Assay

8) Phototoxicity

• In vitro 3T3 NRU Phototoxicity Test

Unintended drug-induced arrhythmia, in particular Torsade de Pointe arrhythmia (TdP), have been responsible for

approximately 21.4% of drug withdrawals from markets between 1990 and 2012. For the past decade, cardiac safety

screening studies have been conducted according to ICH S7B and ICH E14 guidelines. The ICH S7B guideline includes an

in vitro IKr (hERG) assay and an in vivo ECG assay to identify the potential for delayed repolarization (QT interval

prolongation). Although hERG is the most important channel related to the risk of TdP, hERG screening alone cannot

reliably detect potential cardiac adverse side effects. Furthermore, this over-simplified and highly sensitive approach can

result in unwarranted attrition of novel drug candidates owing to false-positive findings. Recently a new paradigm to

examine cardiotoxicity called the Comprehensive in Vitro Proarrhythmia Assay (CiPA) was proposed to replace the

current ICH S7B/E14 guidelines. The CIPA core assays include: 1. the assessment of drug candidate effects on multiple

human ventricular ionic channels and in silico reconstruction of human heart ventricular action potential to predict the

proarrhythmic risk; and 2. the confirmation study using human stem-cell derived cardiomyocytes2,3.

Figure 1. Diagrammatic representation of CiPA2

WuXi AppTec provides a combination of assay platforms that include manual and automated patch-clamp and

microelectrode array (MEA) to access drug candidate effects on multiple cardiac ion channels (Table 1) as well as stem

cell-derived ventricular cardiomyocytes as CIPA recommended.

Table 1. Cardiac ion channel panel

(CIPA recommended3).

Figure 2. Sample traces recorded from QPatch automatic patch clamp system (A), Manual patch clamp (B) and MEA

system (C). The cells used in QPatch are the CHO-hERG stable cell line, while in Manual patch clamp are HEK-Cav1.2

stable cell line; hiPSC-vCMs were used in MEA system.

WuXi AppTec Biology obtains human induced pluripotent stem cell-derived ventricle cardiomyocytes (hiPSC-vCMs) from

a third party vendor. The purity of the ventricular cardiomyocytes is over 90%, which is the highest number seen in the

literature. The action potential, main ion channel properties and development process of the hiPSC-vCMs have all been

fully validated using the manual patch clamp system at WuXi AppTec4. WuXi AppTec Biology now offers CIPA

confirmatory electrophysiological test3 with MEA (Maestro Pro, Axion) to analyze the effects of compounds on hiPSC-

vCMs.

In addition, to satisfy the mechanistic study or high throughput study needs, WuXi AppTec Biology also provides

traditional radioligand binding and fluorescence signal detection assays. The service for some other cardiac targets such

as Kv1.5 (Ikur) and Cav3.2 (ICa,T) is also available.

Cytotoxicity testing is mandated by the FDA and CFDA for IND/CTA submission and is typically performed during the

nonclinical phase of discovery. In recent decades, it is well accepted that in vitro cytotoxicity testing methods should be

considered before animals are dosed to examine acute oral systemic toxicity. Identifying potential cytotoxicity early in

the process can dramatically save both time and capital by eliminating likely toxic compounds prior to pivotal animal

studies. Additionally cell-based assays are easy to scale for medium or high throughput screening and guiding go/no-go

decisions.

Cell Viability: ATP production measured in active cells

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(A) In a 384-well HepG2 cell viability assay,

24 hours of Nefazodone treatment

inhibited cell viability in a concentration

dependent manner (n=5).

(B) After 72 hours treatment in 384-well

plates, Staurosporine showed

concentration dependent inhibition on

the viability of HepG2 cells (n=5).

The CellTiter-Glo® Luminescent Cell Viability Assay (Promega): This is a homogeneous method to determine the

number of viable cells based on quantitation of the ATP present, which is a marker for the presence of metabolically

active cells. WuXi AppTec Biology has fully validated this assay with a wide range of cell types, in both 96- and 384-well

format, using EnVision (Perkin Elmer) as the luminescent signal reader.

Apoptosis: Caspase assay

Activation of the caspase cascade is an integral event in the apoptotic pathway. WuXi AppTec Biology uses Caspase-

Glo® 3/7 Assay kit (Promega) to measure caspase-3 and caspase-7 activities. The assay was fully validated on HepG2

Cells with EnVision as the Luminescence signal reader. Available in both 96- and 384-well format.

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Staurosporine increased caspase activity. In a 96-well plate assay staurosporine

concentration-dependently increased caspase activities in the HepG2 cells. EC50

was 0.07 µM and 0.23 µM for 6-hour and 18-hour staurosporine treatment,

respectively.

CellTox™ Green Cytotoxicity Assay (Promega)

The CellTox™ Green Cytotoxicity Assay measures changes in membrane integrity that occur as a result of cell death. The

assay system uses a proprietary asymmetric cyanine dye that is excluded from viable cells but preferentially stains dead

cell DNA. Viable cells produce no appreciable increases in fluorescence. Therefore, the fluorescent signal produced by

the dye binding to the dead-cell DNA is proportional to cytotoxicity. We now offer this assay in both 96- and 384-well

format on various cell types, using Envision as the signal detector. This assay can be combined with CellTiter-Fluor™ Cell

Viability Assay or CellTiter-Glo® Luminescent Cell Viability Assay (Promega).

CellTox™ Green Dye binds DNA of cells

with impaired membrane integrity.

LN-18 cells were exposed to staurosporine with indicated concentrations in a

384-well assay plate for 24 hours. Fluorescence associated with cytotoxicity

was measured after CellTox™ Green Reagent was applied, then the CellTiter-

Fluor Reagent was applied and fluorescence signal was measured. These

measurements produced similar EC50 values.

ApoTox-Glo™ Triplex Assay (Promega)

The ApoTox-Glo™ Triplex Assay combines three Promega assay chemistries to assess viability, cytotoxicity and caspase

activation events within a single assay well. The first part of the assay simultaneously measures two protease activities;

one is a marker of cell viability, and the other is a marker of cytotoxicity. The second part of the assay uses the Caspase-

Glo® Assay Technology to detect caspase activities. WuXi AppTec has validated this assay with various cell types, using

Envision as the signal reader.

In the 384-well assay with LN-18 cells, seven hours staurosporine treatment

caused concentration-dependent decrease in cell viability, increase in

cytotoxicity and increase in caspase-3/7 activities.

Mitochondria play a pivotal role in cellular energy (ATP) production and maintaining homeostasis. Mitochondrial

dysfunction is increasingly implicated as a major contributor to drug-induced toxicity, leading to the discontinuation of

prominent drugs, including troglitazone, cerivastatin and nefazodone. In addition to post-market drug withdrawals,

mitochondrial liabilities have also been associated with many drugs carrying a black box label for hepatic and cardiac

toxicity.

WuXi AppTec offers a set of in vitro assays to access mitochondrial toxicity from different endpoint.

Mitochondrial Membrane Potential Assay

Mitochondrial membrane potential (MMP) is tightly interlinked to many mitochondrial processes so it is a key indicator

of mitochondrial function and cell health. The dissipation of MMP is considered an early indicator of apoptosis.

WuXi Biology offers a plate based HCS assay to detect the MMP, using the Acumen Cellista (TTP Labtech) with MITO-ID®

Membrane Potential Cytotoxicity Kit (ENZO Life Sciences). The assay is available in both 96- or 384-well format, and in a

wide range of cells.

The MITO-ID® Membrane Potential Cytotoxicity Kit utilizes a cationic dual-emission dye that exists as green fluorescent monomers in the

cytosol, and accumulates as orange fluorescent aggregates in the mitochondria. Cells exhibit a shift from orange to green fluorescence as

mitochondrial function becomes increasingly compromised.

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After one hour treatment, two references compounds, FCCP and Antimycin

A, showed MMP inhibition in the 384-well assay plate using HepG2 cells

(n=4). The IC50 values are close to the literature.

Mitochondrial Reactive Oxygen Species (ROS) Assay: ROS-Glo™ H2O2 Assay

Mitochondrial dysfunction usually causes increased free radical production. The predominant source of free radical

generation is the mitochondrial respiratory chain, and inhibition of this process is often connected to increased levels of

reactive oxygen species (ROS). In the different ROS generated in cell culture, H2O2 is convenient to assay because of the

long half-life in cultured cells. A change in H2O2 can reflect a general change in the ROS level.

WuXi Biology has validated a plate based assay using ROS-Glo™ H2O2 assay kit (Promega) to detect H2O2 levels. The

assay is available in both 96- or 384-well format, and can also be combined with other cytotoxicity measurements.

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CellTiter-Glo Assay

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) In this representative example, the ROS-Glo™ H2O2 assay and the CellTiter-Glo®

Luminescent Cell Viability were performed on the same HepG2 cells in a 384-well

format. The cells were treated with ROS-generating compound menadione as well as

H₂O₂ substrate. After incubation at 37°C for two hours, the half volume of supernatant

was used for ROS-Glo™ H₂O₂ detection, while the cells were lysed for CellTiter-Glo®

detection. The luminescence signal from both assays measured with EnVision.

Menadione had an EC50 of 8.96 μM in the ROS-Glo™ H₂O₂ assay, and an IC50 of 11.28

μM in the CellTiter-Glo® assay.

Oxygen Consumption Assay: MitoXpress® Xtra OCR Assay (HS method) (Luxcel)

The MitoXpress® Xtra assay directly measures immediate and acute drug effects on mitochondrial oxidative

phosphorylation (Oxygen Consumption Rate, OCR) and is the most effective high throughput screen for Mitochondrial

Toxicity using whole cells. Oxygen consumption is the most important parameter for the direct and specific assessment

of the function of the electron transport chain, the cornerstone of oxidative phosphorylation and cellular metabolism. In

the MitoXpress® Xtra OCR assay, the MitoXpress® reagent is quenched by oxygen, whereby oxygen depletion caused

by mitochondrial activity causes an increase in probe signal, with rates of oxygen consumption calculated from the

changes in fluorescence signal over time. Available in a 384- or 96-well high throughput format, this assay is compatible

with a very wide range of primary, iPS or cell line models and both 2D and 3D culture systems.

Results from a typical 96-well MitoXpress® Xtra (HS method)

OCR concentration-response assay for two mitochondrial

inhibitors, Rotenone and Nefazodone. The HL60 cells were

used in the assay.

MitoXpress® Cellular Energy Flux OCR/ECAR Assay (Luxcel)

A deeper, investigative analysis to understand the mechanism of mitochondrial toxicity and its relationship to cellular

ATP production is made possible through the addition of Luxcel Biosciences pH Xtra – Glycolysis Assay; detecting in real-

time the combined effects on OCR and extracellular acidification rate (ECAR)5. This assay is available in a 384- or 96-well

high throughput format.

A BResults from typical 384-well MitoXpress® Cellular

Energy Flux OCR/ECAR assay with HL60 cells, illustrating

the difference between a classic mitochondrial inhibitor,

Rotenone, which decreased OCR / increased ECAR (A);

and a non-specific cytotoxic drug, Tamoxifen, which

decreased both OCR and ECAR (B).

Glucose/Galactose Assay

Replacing glucose with galactose in the cell media increases the reliance of the cells on mitochondrial oxidative

phosphorylation, thereby increasing susceptibility to the implications of mitochondrial insult. By comparing the

differential toxic effects on glucose and galactose grown cells it will differentiate mitochondrial toxicity from non-specific

cytotoxicity.

We use HepG2 cells cultured in either glucose (25 mM) or galactose (10 mM), with cytotoxicity assessed using CellTitre-

Glo™ (Promega). A mitochondrial toxicant is indicated by a greater than three-fold change in IC50 value observed in the

glucose media compared to the galactose media.

Illustration of the 24-hour treatment data for the

mitochondrial uncoupler, FCCP (A), and inhibitor,

nefazodone (B). A > 3-fold and 3.7-fold increase in

IC50 value is observed for FCCP and nefazodone,

respectively, in glucose media compared with

galactose media.

MitoBiogenesis Assay

Determination of the mitochondrial biogenesis level relative to the cellular protein synthesis provides important

information on potential mitochondrial toxicity. This is particularly important for antiviral and antibiotic new drug

development because the similarity between mitochondrial biogenesis and bacterial/viral replication. Many such drugs

can cause serious mitochondrial toxicity.

Our mitobiogenesis assay uses Odyssey (LI-COR) with MitoBiogenesis™ In-Cell ELISA Kit (IR) (Abcam). The assay has been

validated on HepG2 cells and are available in both 96- and 384-well format.

This assay simultaneously measures the levels of two

mitochondrial proteins, Mitochondrial DNA encoded

COXI and nuclear DNA encoded SDH-A. The specific

inhibition of Mitochondrial DNA encoded protein

synthesis by chloramphenicol is thus easily observed.

Inhibition of mitochondrial biogenesis by chloramphenicol.

(A) HepG2 were seeded at 1200 cells/well in 384 well plate,

Chloramphenicol inhibits COX-I protein synthesis relative to

SDH-A protein synthesis. (B) The overall mitochondrial

biogenesis inhibition was calculated from the ratio of

measured COX-I/SDH-A protein levels.

Phospholipidosis is a lysosomal storage disorder and characterized by the accumulation of excess phospholipid

complexes within the internal lysosomal membranes. Cationic amphiphilic drugs (CADs), such as antibiotics,

antidepressants, antihistamines and other prescription drugs, have been identified as inducers of phospholipidosis. The

US FDA has acknowledged that drug-induced phospholipidosis is an adverse drug reaction7.

Steatosis is the situation of cytoplasmic accumulation of neutral lipids. Some drug can interfere with hepatic lipid

processing, leading to accumulation of triglycerides within the liver cells. This condition may lead to harmful liver

inflammation, or steatohepatitis.

Both drug induced phospholipidosis and steatosis are often reversible conditions without remarkable consequences;

however, after prolonged exposure to a particular drug, they can lead to long-term toxic effects. Therefore drug induced

cellular lipotoxicity leading to phospholipidosis and/or steatosis should be evaluated during the early drug discovery

stage to minimize potential risk.

WuXi AppTec now offer in vitro HCS assays on HepG2 cells using the HCS LipidTOX™ Stains (Thermo Fisher Scientific),

with the CQ1 (confocal quantitative image cytometer, Yokogawa Electric Corporation), or Acumen Cellista (TTP Labtech)

as the image reader. Phospholipidosis is detected with the LipidTOX™ Red phospholipid stain, while steatosis is detected

with the LipidTOX™ Green neutral lipid stain.

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HepG2 cells were exposed to test articles and LipidTox reagent in a 96-well plate for 48 hours. Hoechst 33342 nuclear staining was used as

control. Fluorescence images were captured with CQ1. LipdTox red phospholipidosis and Hoechst 33342 emit red and blue fluorescence in the

same field, respectively.

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Results for red fluorescence were normalized to those of blue fluorescence

(Hoechst). Propranolol concentration-dependently increased phospholipid

staining.

Steatosis assay

HepG2 cells were exposed to the test articles in 96-well plate for 48 hours. Fixed cells were stained with LipidTOX™ Green neutral lipid stain

and the Hoechst 33342. Fluorescence images were captured with CQ1. The Green Neutral Lipid Stain and Hoechst 33342 emit green and blue

fluorescence in the same field, respectively.

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Results for green fluorescence (LipidTox Green Neutral Lipid Stain) were

normalized to those of blue fluorescence (Hoechst). Both Cyclosporin A and

Loratadine concentration-dependently increased the Green Neutral Lipid

Stain, suggesting the accumulation of steatosis.

The liver plays a central role in transforming and clearing chemicals and is susceptible to the toxicity from these agents.

Drug-induced liver injury (DILI) is caused by many hundreds of widely prescribed drugs and is a leading cause of drug

development and registration failure, withdrawal of approved drug, and cautionary labeling which restricts drug usage.

The assessment of compound-induced hepatotoxicity has traditionally relied on in vivo testing; however the studies are

limited to a small amount of late stage compounds. In addition there are species-specific differences. Since most severe

DILI is due to hepatocellular injury, in vitro hepatotoxicity testing would provide valuable information to predict potential

risk.

WuXi Biology offers multiplexed in vitro assays to assess potential hepatotoxicity at cellular level. The assays use HepG2

(human liver hepatocellular carcinoma) cells, or human primary hepatocytes. By using these cells as in vitro models, all

assays listed in the General Cytotoxicity, Mitochondrial Toxicity, and Lipotoxicity sections in this brochure can be used to

evaluate potential hepatotoxicity. The combination of these assays will provide valuable information from different

endpoint to analyze the mechanism and severity of hepatotoxicity.

Recommended HepG2 hepatotoxicity first tier assays:

• Cell Viability Assay: a very sensitive marker to detect general toxicity;

• Mitochondrial Membrane Potential Assay: an indicator of poor respiratory capacity and cell health;

• Mitochondrial Reactive Oxygen Species (ROS) assay: ROS increase may result in significant cell structure

damage and cause oxidative stress;

• Caspase assay: measuring Caspase-3/7 activities.

Assay Features and Advantages:

• Plate-based assay (96- or 384-well plate) read by plate reader or high content image reader;

• Medium to high throughput with high sensitivity;

• Low cost with a short turnaround time;

• Short term (24 h) and long term (10~14 d) toxicity assays available.

HepG2 Cell Viability Assay

Reference

CompoundStaurosporine Tamoxifen Nefazodone Rotenone

Treatment 72h 72h 24h 24h

IC50 (µM) 0.036 ± 0.007 10.55± 0.59 48.09± 7.11 4.49± 1.05

n n=5 n=5 n=5 n=5

IC50 values of several reference compounds in our HepG2

cell viability assay.

The kidney’s primary function is the filtration and excretion of soluble waste while retaining key biochemicals. Thus its

design as a selective filter makes it particularly susceptible to toxic injury. Renal tubular cells, in particular proximal tubule

cells, are especially vulnerable to drug toxicity due to their prominent role in the filtration process which exposes them to

high levels of circulating toxins. Thus, our assays use human-derived cellular systems originating from the renal proximal

tubule epithelial cell line (HK-2) to maximize the predictive power of nephrotoxicity. The HK-2 cells are derived from

normal adult human kidneys and immortalized by transduction of the E6/E7 genes via HPV- 6.

WuXi AppTec has developed several nephrotoxicity assays to enable high throughput screening providing an early

assessment of nephrotoxic effects and the potential for kidney-specific cellular injury. In general, all assays listed in the

General Cytotoxicity and Mitochondrial Toxicity sections above can be used to assess nephrotoxicity, including the Cell

Viability Assay, Apoptosis assay, etc.

Validation data on HK-2 cells using the CellTiter-Glo viability assay. The assay

was run at 9 concentrations in triplicate and the dose response curve of

paclitaxel is presented. Two reference compounds, paclitaxel and staurosporine

were tested. The IC50 were 20 nM and 16 nM, respectively.

When DNA is exposed to particular chemicals, mutations and other damage can occur leading to cancer and/or

teratogenic effects. The severity of these effects then necessitates examining whether new or existing chemicals intended

for human use have any effect on DNA. This genotoxic potential is an integral part of the basic toxicological information

package used in the decision-making and risk assessment process of drug development. Since no single test is capable

of detecting all relevant genotoxic endpoints, a battery of tests for genotoxicity is recommended by regulatory agencies.

Mini Ames AssayThe mini Ames assay is modified from the standard Ames test that uses 6-well plates and 20% of the typical Ames assay

medium. The purpose is to generate a rapid screening test that utilizes small quantities of the test compound but still

gives results in agreement with those of the standard Ames assay. The mini Ames assay is widely used as an early

compound screen during lead optimization or preclinical candidate selection.

WuXi offers the Ames Microplate format (MPF) Assay, which corresponds to the Ames Fluctuation Assay that is cited in

the guidelines of OECD and FDA. The MPF assay uses a liquid format and 384-well microplates, which requires less test

compound, is time and cost-effective, but still shows good correlation with the traditional plate incorporation assay. The

MPF assay employs four Salmonella typhimurium TA98, TA100,TA1535 and TA1537, E. coli strain wp2 uvrA or wp2 uvrA

[pKM101] for mutagenic potential investigation in the presence or absence of a metabolic activation system (e.g., Aroclor

1254-induced rat liver S9). It generates a rapid screening and increases the throughput for drug mutagenic potential

assessment in the early stage.

Bacteria are preincubated with the test compound (6 concentrations, a positive and a negative control) in exposure medium supplied with

sufficient histidine or tryptophan with or without S9 for 90 minutes. The culture is then diluted into pH indicator medium without histidine (for

S. typhimurium) or tryptophan (for E. coli ) and aliquoted into 48 wells of a 384-well plate. After 48 h incubation, cells undergoing the reversion

to prototrophy will change the medium colour from purple to yellow, which can be detected visually or by microplate reader.

Relative mutagenic potential of reference compounds as detected

by Salmonella typhimurium TA98 and TA100. The number in the

table indicates the revertants/48 wells.

In vitro Micronucleus Assay

The in vitro micronucleus assay is another part of the recommended regulatory testing battery for genotoxicity as an

alternative to the chromosomal aberration assay. This assay also involves the analysis of chromosomal irregularities, but

unlike the chromosomal aberration assay which requires considerable training, micronucleus testing is much simpler,

faster and scalable. The micronucleus test examines the presence of micronuclei in the cytoplasm during interphase.

Micronuclei may originate from acentric chromosome fragments (i.e. lacking a centromere), or whole chromosomes that

are unable to migrate to the poles during the anaphase stage of mitosis.

In Vitro 3T3 NRU Phototoxicity Test

The in vitro 3T3 NRU phototoxicity test is used to identify the phototoxic potential of a test substance induced by the

excited chemical after exposure to light. The test evaluates photo-cytotoxicity by the relative reduction in viability of cells

exposed to the chemical in the presence versus absence of light. Now the test has been fully validated in WuXi Biology

Group using the permanent mouse fibroblast cell line, Balb/c 3T3, following the protocol documented in the OECD (432)

guideline. SOL 500 (Dr. Hönle AG) was used as the light source while SpectraMax2 (Molecular Devices) plate reader was

used to collect data.

Phototoxicity is defined as a toxic response from a substance applied to the body, which is either elicited or increased

after subsequent exposure to light, or that is induced by skin irradiation after systemic administration of a substance. The

regulatory guidelines for phototoxicity are covered by the ICH S108, and the OECD Guideline for the Testing of Chemicals

432: In Vitro 3T3 NRU Phototoxicity Test9.

Enabling Unbounded Possibilities

Contact

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± ±

Results of two phototoxic compounds, chlopromazine and amiodarone hydrochloride. For the result evaluation, the Photo-Irritation-Factor (PIF)

was calculated using the following formula: PIF = IC50(-UV)/IC50(+UV). In this study the PIF values of the two compounds are consistent with the

reference data in OECD (432) guideline. A test material is defined as having a potential phototoxic hazard if the PIF value is or greater than 5.

References:

1. NRC Toxicity Testing in the 21st Century: A Vision and a Strategy. Washington, DC: The National Academies Press (2007).

2. Sager, et al., Rechanneling the cardiac proarrhythmia safety paradigm: A meeting report from the cardiac safety research

consortium. American Heart Journal, 167: 292–300 (2014).

3. Colatsky, et al., The Comprehensive in Vitro Proarrhythmia Assay (CiPA) initiative —Update on progress. J Pharmacol

Toxicol Methods, 81: 15-20 (2016).

4. Pei, et al., Chemical-defined and albumin-free generation of human atrial and ventricular myocytes from human

pluripotent stem cells. Stem Cell Res, 19: 94-103 (2017).

5. Sakamuru, et al., Application of a homogenous membrane potential assay to assess mitochondrial function. Physiological

Genomics, 44(9): 495–503 (2012).

6. Hynes, et al., A high-throughput dual parameter assay for assessing drug-induced mitochondrial dysfunction provides

additional predictively over two established mitochondrial toxicity assays. Toxicology in Vitro, 27(2): 560–569 (2013)

7. Chatman, et al., A Strategy for Risk Management of Drug-Induced Phospholipidosis. Toxicol Pathol, 37(7): 997-1005 (2009).

8. ICH Harmonised Tripartite Guideline: Photosafety Evaluation of Pharmaceuticals S10. (2013).

9. OECD Guideline for the Testing of Chemicals 432: In Vitro 3T3 NRU Phototoxicity Test. (2004).